Abstract
Purpose :
Photoreceptor (PR) metabolism is unique among other neurons in that they primarily process glucose into lactate via aerobic glycolysis. This unique adaptation allows maintenance of the highly-specialized PR cell structure under basal conditions and provides a survival advantage during prolonged nutrient deprivation. The main drivers of aerobic glycolysis, HK2 and PKM2, are expressed primarily in PRs in the retina. We have previously demonstrated that the conditional knockout (cKO) of HK2 from rods sensitizes PRs to cell death following retinal detachment whereas cKO of PKM2 protects PRs from cell death during acute nutrient deprivation. Here we present data from animals lacking both HK2 and PKM2 in rod PRs.
Methods :
Mice harboring a floxed Hk2 allele were crossed to mice carrying a floxed Pkm2 allele. Homozygotes for both floxed genes were generated and crossed to animals harboring a cre recombinase gene under the control of the rhodopsin promoter to create animals with conditional deletion of both Hk2 and Pkm2 from rods specifically. OCT was performed monthly and ERG was performed at specific time points. Whole eyes were fixed,and sectioned for H&E staining. The total number of PRs at each time point were counted using a custom macro in ImageJ and normalized to inner retinal area. Immunofluorescence was used on sections to verify deletion of HK2 and PKM2 from rods. Whole retinas were extracted for protein, RNA, or metabolite purification for Western blotting, qRT-PCR, or metabolomics analysis, respectively. Experimental retinal detachment was induced and whole eyes were collected at 3 days for TUNEL analysis and at 1 month for PR viability assessment.
Results :
OCT analysis showed shortening of outer segments by 4 months of age and thinning of the outer nuclear layer (ONL) by 5 months of age. Western blotting and immunofluorescence confirmed successful deletion of both HK2 and PKM2 from rods and a compensatory upregulation of HK1 and PKM1. Total cell counts of H&E stained retina showed a significant loss of photoreceptors at 10 months of age compared to control animals.
Conclusions :
Our data demonstrate that loss of both HK2 and PKM2 in double cKO retinas leads to outer segment shortening followed by PR degeneration, a phenotype different from the conditional deletion of HK2 or PKM2 alone. Early shortening of outer segments suggests metabolic, structural, and functional deficits prior to loss of PRs.
This is a 2021 ARVO Annual Meeting abstract.